IMPORTANCE OF THE LOOP CONNECTING A-HELIX AND B-HELIX OF HUMAN INTERFERON-GAMMA IN RECOGNITION BY INTERFERON-GAMMA RECEPTOR

Characterization of murine human hybrid interferon-gamma (IFN-gamma) m olecules suggests that substitution of the peptide connecting the A an d B helices in human IFN-gamma with the murine sequence significantly blocks the protein's binding to the human interferon-gamma receptor. M utagenesis showed that this effect is localized to the central part of this A-B loop peptide, particularly Ser(20), Asp(21), Val(22), and Al a(23). One mutant, IFN-gamma/A23E,D24E,N25K, was examined by NMR. This ''EEK'' mutation does not significantly alter the conformation of int erferon-gamma, suggesting that the effects of these mutations are not the result of global conformational changes. The A-B loop is near hist idine 111, a residue previously shown to be important in receptor-liga nd interaction (Lunn, C.A., Fossetta, J., Dalgarno, D., Murgolo, N., W indsor, W., Zavodny, P.J., Narula, S.K., and Lundell, D. (1992) Protei n Eng. 5, 253-257). We show that copper forms a complex between histid ine 19 in the A-B loop and histidine 111. This metal complex lacks the ability to interact with the interferon-gamma receptor. These results suggest that the A-B loop contains important struc tural information needed for receptor-ligand binding and hence biological activity of hu man interferon-gamma.